SEROEPIDEMIOLOGICAL SCREENING OF LEISHMANIA INFECTION IN DOGS AND CATS: THE ROLE OF THE VETERINARIAN IN A HIGHLY ENDEMIC AREA IN SICILY

BACKGROUND Leishmaniosis is a zoonotic disease caused by Leishmania spp. with a wide spectrum of clinical signs, lymphadenomegaly, skinocular lesions, weight loss, signs of renal failure [1,2]. Many subclinical cases of leishmaniasis without illness could play an important role in the maintenance of Leishmania infection in endemic areas. Veterinary surveillance of dogs/cats owners could help to update the seroprevalence status of Leishmania infantum infection in a random court of sick and apparently healthy dogs and cats from western Sicily (Bagheria and neighboring areas) using indirect fluorescent antibody test (IFAT). MATERIAL AND METHODS A total of 268 dogs and 9 cats living in western Sicily were randomly sampled during the 2020 year. Specific antibodies to L. infantum were detected using the IFAT against in-house cultured promastigotes. Leishmania strain was used as an antigen fixed on multispot microscope slides. The feline and canine sera were both prepared by serial 2-fold dilutions (1:40 to 1:5120) in phosphate buffered saline and added to the antigen-coated wells. Fluorescent anti-cat/anti-dog immunoglobulin G antibody was used for detection. CONCLUSION Western Sicily is an active focus for canine and feline leishmaniosis in the Mediterranean area. The results of the present study indicate a high exposure rate to Leishmania (about 33% dogs and 44% cats result positive or suspected) in a random population, suggesting that they are infected with L. infantum. Moreover, 5 dogs and 1 cat previously classified as apparently healthy were seropositive with a titer ≥1:160. In conclusion, veterinary surveillance of dogs/cats could help to control the increase of L. infantum infections, especially in areas of high endemicity. RESULTS 49/268 (18.3%) sampled dogs tested positive to IFAT with a titer ≥1:160 and 40/268 (15%) tested suspected (titers 1:40-1:80), for L.infantum infection (Table 1). An additional 2/9 (22.2%) cats were seropositive with a titer of 1:160 and 2/9 (22.2%) cats were seropositive with a titer of 1:80 (Table 1). Figure 1 shows geographical distribution of L. infantum seroprevalenc in dogs/cats sampled from western Sicily. CONCLUSION Western Sicily is an active focus for canine and feline leishmaniosis in the Mediterranean area. The results of the present study indicate a high exposure rate to Leishmania (about 33% dogs and 44% cats result positive or suspected) in a random population, suggesting that they are infected with L. infantum. Moreover, 5 dogs and 1 cat previously classified as apparently healthy were seropositive with a titer ≥1:160. In conclusion, veterinary surveillance of dogs/cats could help to control the increase of L. infantum infections, especially in areas of high endemicity

SEROEPIDEMIOLOGICAL SCREENING OF LEISHMANIA INFECTION IN DOGS AND CATS: THE ROLE OF THE VETERINARIAN IN A HIGHLY ENDEMIC AREA IN SICILY

Western Sicily is an active focus for canine and feline leishmaniosis in the Mediterranean area. The results of the present study indicate a high exposure rate to Leishmania (about 33% dogs and 44% cats result positive or suspected) in a random population, suggesting that they are infected with L. infantum. Moreover, 5 dogs and 1 cat previously classified as apparently healthy were seropositive with a titer ≥1:160. In conclusion, veterinary surveillance of dogs/cats could help to control the increase of L. infantum infections, especially in areas of high endemicity

"Stilbene ST18, terfenile TR4 e carrier innovativi: effetti in cellule di Leishmania spp. ed in modelli sperimentali in vivo".

Convegno Nazionale I RISULTATI DELL’ATTIVITÁ DI RICERCA DELL’IZS SICILI

EVALUATION OF RPMI-PY MEDIUM FOR TRYPANOSOMA CRUZI AND DIFFERENT LEISHMANIA SPECIES

Trypanosoma spp. and Leishmania spp. are causal agents of a number of parasitic diseases. Culture media can be divided into 3 main categories: semisolid, biphasic, and liquid. While biphasic and semisolid culture media need blood, an important factor for the reproduction of parasites, most liquid media require fetal calf serum or erythrocyte lysate. A culture media RPMI-PY demonstrated a good performance in terms of time and parasitic load of L. infantum compared to other culture media. The aim of the work was to evaluate the performance of RPMI-PY medium in different Leishmania species and also to evaluate in T. cruzi culture. RPMI-PY is likely to be valuable additions to laboratory practice in light of the relatively simple recipes, general availability of the components, and in terms of suitability because rabbit breeding is not necessary and the costs are lowered and can be used for all Leishmania species and to cultivate T. cruzi

STILBENE ST18 AND TERFENYL TR4: IN VITRO ACTIVITY AGAINST TRYPANOSOMA CRUZI

Chagas disease is caused by the parasite Trypanosoma cruzi, which is transmitted to animals and people by insect vectors that are found only in the Americas. Chagas disease and Leishmaniasis are life-threatening illnesses caused by the protozoan parasites Leishmania spp. and Trypanosoma cruzi, respectively. They are known as “neglected diseases” due to the lack of effective drug treatments and the scarcity of research work devoted to them. Therefore, the development of novel and effective drugs is an important and urgent need. Natural products are an important source of bioactive molecules for the development of new drugs. Recently, studies showed an interesting cytotoxic action of Stilbene ST18 and the Terphenyl TR4 compounds in Leishmania. In this study, we evaluated the in vitro trypanocidal activity of ST18 and TR4 and Nifurtimox, drug used for the treatment of Chagas disease. In addition, we evaluated the compounds action in infected macrophages with Trypanosoma cruzi. Results showed that three compounds exhibited significant activity against Trypanosoma compared to Nifurtimox. ST18 and TR4 compounds inhibited Trypanosoma growth with IC50 values of 4.5 and 32 μM, respectively. The treatment of infected macrophages with trypanosomes with the IC90 compounds showed a reduction of infection compared to control: ST18 reduced the infected cells to 52 %, TR4 reduced the infected cells to 63%. In conclusion, these news compounds could be considered as promising lead drugs for the development of new therapies for the treatment of Chagas disease

The host micro-RNA cfa-miR-346 is induced in canine leishmaniasis

none8noBackground Leishmaniases are a group of anthropo-zoonotic parasitic diseases caused by a protozoan of the Leishmania genus, affecting both humans and other vertebrates, including dogs. L. infantum is responsible for the visceral and occasionally cutaneous form of the disease in humans and canine leishmaniasis. Previously, we have shown that L. infantum induces a mild but significant increase in endoplasmic reticulum (ER) stress expression markers to promote parasites survival in human and murine infected macrophages. Moreover, we demonstrated that the miRNA hsa-miR-346, induced by the UPR-activated transcription factor sXBP1, was significantly upregulated in human macrophages infected with different L. infantum strains. However, the ER stress response in infected dogs, which represent an important reservoir for Leishmania parasite, was described once recently, whereas the miR-346 expression was not reported before. Therefore, this study aimed to investigate these pathways in the canine macrophage-like cell line DH82 infected by Leishmania spp. and to evaluate the presence of cfa-miR-346 in plasma of non-infected and infected dogs. The DH82 cells were infected with L. infantum and L. braziliensis parasites and the expression of cfa-mir-346 and several ER stress markers was evaluated by quantitative PCR (qPCR) at different time points. Furthermore, the cfa-miR-346 was monitored in plasma collected from non-infected dogs (n = 11) and dogs naturally infected by L. infantum (n = 18). Results The results in DH82 cells showed that cfa-mir-346 was induced at both 24 h and 48 h post-infection with all Leishmania strains but not with tunicamycin, accounting for a mechanism of induction independent from sXBP1, unlike what was previously observed in human cell lines. Moreover, the cfa-miR-346 expression analysis on plasma revealed a significant increase in infected dogs compared to non-infected dogs. Conclusions Here for the first time, we report the upregulation of cfa-miR-346 induced by Leishmania infection in canine macrophage-like cells and plasma samples of naturally infected dogs. According to our results, the cfa-miR-346 appears to be linked to infection, and understanding its role and identifying its target genes could contribute to elucidate the mechanisms underlying the host–pathogen interaction in leishmaniasis.Buffi, Gloria; Diotallevi, Aurora; Ceccarelli, Marcello; Bruno, Federica; Castelli, Germano; Vitale, Fabrizio; Magnani, Mauro; Galluzzi, LucaBuffi, Gloria; Diotallevi, Aurora; Ceccarelli, Marcello; Bruno, Federica; Castelli, Germano; Vitale, Fabrizio; Magnani, Mauro; Galluzzi, Luc

FELINE LEISHMANIASIS: SEROLOGICAL AND MOLECULAR DETECTION OF AN EMERGENT DISEASE IN A NON-ENDEMIC AREA OF NORTHERN ITALY

In recent decades feline leishmaniosis (FeL) has become an emerging disease, also in non-endemic areas for the canine infection. This study updates the epidemiological status for FeL in cats in northern Italy and compares results with previous studies of the same feline population. Co-infections with feline retroviruses FIV and Field were also investigated. Stray, shelter and owned cats from different cities in the Lombardy region of northern Italy, were prospectively randomly sampled between January 2020 and May 2021. A total of 255 cats were tested for L. infantum: 240/255 for antibodies by IFAT and 234/255 and 198/255 for Leishmania DNA by PCR on whole blood and lymph nodes, respectively. Rapid ELISA test was used to detect FIV or FeLV infection. Overall, 26/255 (10.2%) cats tested positive for L. infantum: in 8/26 cats Leishmania DNA was found in popliteal lymph nodes (Leishmania/ml range from 15 to 60), 6/26 were PCR positive on whole blood (Leishmania/ml range from 5 to 80) and 15/26 IFAT seropositive at titers ranging from 1:80 to 1:320. Two Leishmania infected cats were also FIV+FeLV coinfected, another was FIV positive and one was FeLV positive. A high prevalence of FeL was found in a non-endemic area of northern Italy, with an increasing trend in infection rates

Microsatellite panel definition to characterize Leishmania strains isolated from human samples in an italian endemic region

The Leishmaniasis affects people, domestic and wild animals in temperature, subtropical and tropical regions. The natural cycle involves phlebotominae sandfly vectors transmitting the parasite to the vertebrate host. The insects influence the epidemiology of the disease by their geographical distribution in the seasons and the specific vectorial capacity. Human Leishmania infections are increasing every year in Sicily, which represent the region with the highest endemic level of the disease in Italy. Among different approaches employed for the diagnostic the parasites isolation remains the gold standard

Zoonotic infectious diseases in transplanted immunocompromised patients

Background. Immunocompromised patients, like transplant recipients, are a particularly vulnerable group being at higher risk of developing several infectious diseases. Among them, zoonotic diseases, such as visceral leishmaniasis, bartonellosis, Q fever and leptospirosis are a growing concern in immunosuppressed patients as they are more susceptible to develop severe symptoms of the diseases. Objectives. The study aimed at the detection of Leishmania infantum, Bartonella spp., Leptospira spp. and Coxiella burnetii DNA in immunocompromised hosts through molecular methods

Evaluation of a kDNA-Based qPCR Assay for the Detection and Quantification of Old World Leishmania Species

none10noThe parasite protozoan Leishmania, the causative agent of leishmaniasis, includes two subgenera of medical interest: Leishmania (Leishmania) and Leishmania (Viannia). Parasite species detection and characterization is crucial to choose treatment protocols and to monitor the disease evolution. Molecular approaches can speed up and simplify the diagnostic process. In particular, several molecular assays target the mitochondrial DNA minicircle network (kDNA) that characterizes the Leishmania genus. We previously proposed a qPCR assay targeting kDNA, followed by high resolution melt (HRM) analysis (qPCR-ML) to distinguish L. (L.) infantum and L. (L.) amazonensis from L. Viannia species. Successively, this assay has been integrated with other qPCR assays, to differentiate L. (L.) infantum, L. (L.) amazonensis and L. (L.) mexicana. In this work, we tested the applicability of our qPCR-ML assay on L. (L.) donovani, L. (L.) major, L. (L.) tropica and L. (L.) aethiopica, showing that the qPCR-ML assay can also amplify Old World species, different from L. (L.) infantum, with good quantification limits (1 × 10-4-1 × 10-6 ng/pcr tube). Moreover, we evaluated 11 L. (L.) infantum strains/isolates, evidencing the variability of the kDNA minicircle target molecules among the strains/isolates of the same species, and pointing out the possibility of quantification using different strains as reference. Taken together, these data account for the consideration of qPCR-ML as a quantitative pan-Leishmania assay.openCeccarelli, Marcello; Buffi, Gloria; Diotallevi, Aurora; Andreoni, Francesca; Bencardino, Daniela; Vitale, Fabrizio; Castelli, Germano; Bruno, Federica; Magnani, Mauro; Galluzzi, LucaCeccarelli, Marcello; Buffi, Gloria; Diotallevi, Aurora; Andreoni, Francesca; Bencardino, Daniela; Vitale, Fabrizio; Castelli, Germano; Bruno, Federica; Magnani, Mauro; Galluzzi, Luc